Project Summary/Abstract This application targets the unmet medical needs of patients with bile duct paucity who often develop severe cholestatic liver injury that currently is only curable by liver transplantation. Our application builds on our finding that hepatocytes can form intrahepatic bile ducts (IHBDs) that function to reverse cholestasis in a mouse model of severe IHBD paucity. Conversion of hepatocytes-to-cholangiocytes has been reported, but our mouse model establishes that hepatocytes can build a therapeutically effective biliary system from scratch. We hypothesize that our mouse model has revealed the full potential of hepatocyte-to-cholangiocyte conversion because of the severity of its IHBD paucity and its unique genetic makeup, affecting both NOTCH and TGFbeta signaling, the main regulators of bile duct development. We propose to identify the mechanisms responsible for spontaneous IHBD restoration from hepatocytes in our mouse model with the long-term goal of enlightening tissue engineering approaches and to develop a therapy for diseases associated with bile duct paucity. In Aim 1 we will define the interplay between NOTCH and TGFbeta driving conversion of hepatocytes- to-cholangiocytes and assembly into IHBDs. For this we will use in vivo mouse models to perform pharmacologic and genetic approaches modulating TGFbeta signaling for examining the ?steps? (conversion and morphogenesis) of hepatocyte-derived de novo IHBD formation. In addition, we will investigate the mechanism by which loss of a TGFbeta effector influences the association of transcriptional cis-regulatory complexes to induce hepatocyte-to-cholangiocyte transdifferentiation. Aim 2, in which we will define the transcriptional network driving conversion of hepatocytes-to-cholangiocytes and assembly into IHBDs will also inform these efforts. For this we will investigate in converting hepatocytes in vivo which transcription factors are active and validate their efficacy in primary hepatocytes stabilized in a micropatterned co-culture system and a cholangiosphere morphogenesis assay. Using the insight gained from these experiments, we will express effectors of biliary differentiation and tube formation in vivo using non-integrating, nontoxic adeno-associated viral vectors for gene delivery to hepatocytes. Our research will generate new insight into the molecular regulation of hepatic cell identity, biliary development and regeneration.